Chromatin is a complex of DNA and protein, the material of which the chromosomes are made. Chromatin undergoes various structural changes during a cell cycle. In order to investigate the complexes and understand their function, ChIP-Seq was the method of choice to map global DNA binding sites precisely for any protein of interest.
Improved Method to Study Protein-DNA Interactions
CUT&RUN, which stands for cleavage under targets and release using nuclease offers a new approach to pursue epigenetics1. CUT&RUN overcomes various downfalls of ChIP-Seq with improved workflow. CUT&RUN-sequencing has the advantage of being a simpler technique with lower costs due to the high signal-to-noise ratio, requiring less depth in sequencing. CUT&RUN has the potential to replace all ChIP-based applications.
Advantages of CUT&RUN
- No Crosslinking, the factor-bound DNA that is cleaved on both sides of the bound particle is efﬁciently released into solution
- Limited digestion of chromatin only around target sites minimizes fragments
- Better signal to background noise ratio
- Greatly reducing sequencing depth
- Application viable with limited sample amount
CUT&RUN - Combining Perks of ChIP‐Seq Variations
As CUT&RUN is performed on intact cells or nuclei without fragmentation, it can be used to probe all genomic compartments. The cleaved chromatin complexes diffuse out of the nuclei where they can be harvested in supernatant. The rest of the undigested genome is retained in the intact nuclei (Fig 4). For ChIP-Seq on the other hand, the majority of chromatin is sheared or digested resulting in comparison in a worse signal-to-noise ratio (Fig 1-3). Consequently, ChIP-Seq requires more sequencing depth and with each sequencing run additional sample amount, labour time and money.
The CUT&RUN is straightforward and can be completed in under a day using standard lab equipment. CUT&RUN is suitable for application to 100 cells for profiling H3K27me3 or 1000 cells for CTCF sequence-specific DNA- binding protein1. Therefore, CUT&RUN enables targeted genome-wide maps of protein- DNA interactions even for rare cell types. One of the strengths of CUT&RUN is that the entire reaction is performed in situ.
In combination with a proximity ligation assay the generation of factor-specific high resolution maps of nuclear architecture is possible.
Importance of Antibody Selection
The successful execution of cleavage under target and release using nuclease hinges on antibody selection. A factor- or histone-specific antibody is bound to chromatin in situ followed by binding to the antibody of a protein A-micrococcal nuclease (pA-MNase) fusion. As is the case with ChIP, the success depends in large part on the affinity of the antibody for its target and its specificity under the conditions used for binding. The following list serves as an example, Brahma and Henikoff successfully utilized this compilation in 20182.
- Negative Control: Non-Specific Rabbit IgG Antibody ABIN101961
- Rabbit polyclonal Histone H2B Antibody ABIN6579169
- Histone 3 (H3) (3meLys36) Antibody ABIN2668403
- Histone 3 (H3) (3meLys4) Antibody ABIN2668472
- Rabbit polyclonal Anti-Acetyl-Histone H4 AB
- Rabbit polyclonal Anti-Histone H2A.Z Antibody
- Mouse monoclonal Anti-FLAG M2 Antibody
Suggested reagents based on Brahma et al.
- Hypotonic Lysis to release Nuclei
- Imobilize Nuclei with Magnetic Beads
- Incubate with Antibody against PoI
- Incubate with ProteinA-Mnase
- Add Ca+2 (Reaction Start)
- Add Chelator (Reaction Stop)
- Pellet oligonucleosome
- Peter J. Skene and Steven Henikoff (2018): "CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers”. Nature, Volume 13, pages 1006–1019. [PMID: 25652980]
- Sandipan Brahma and Steven Henikoff (2018): "RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast". Mol Cell, Volume 73, Issue2, P238-249. [DOI]